The main objective of this proposal is to isolate and characterize a variety of conditional lethal temperature-sensitive DNA synthesis mutants of the hamster fibroblast continuous cell line BHK 21 to gain knowledge of the mechanism and regulation of DNA replication in mammalian cells. First it will be necessary to modify available mutant selection techniques to render them more highly specific for the acquisition of the class of mutants necessary to carry out the objectives of this proposal. Several approaches will be implemented for the characterization of the acquired mutants: 1) physiological and biochemical analyses in vivo and in vitro; 2) genetic studies by use of the techniques of somatic cell hybridization; and 3) studies using virus as probes to locate defective functions. The first application of these mutants will be for the understanding of functions involved in the synthesis of DNA and its regulation in somatic mammalian cells. However, in view of the extreme importance of this cellular function and of the usefulness of conditional lethal type mutations, these mutants should be valuable tools for investigations in many other areas, such as: 1) studies of somatic cell genetics; 2) studies of the cell cycle in regard to the coordination of DNA replication with growth and cell division; 3) studies of virus-cell interactions as related to the control of DNA replication in normal cells and in malignant cells transformed by oncogenic viruses.